Membrane transport systems are involved in a large number of fundamental cellular activities, and yet most are poorly characterized at the molecular level. The Glut1 glucose transporter is the prototype facilitative transport protein. Facilitative glucose transport is a critical function carried out by virtually all mammalian cells. This process is mediated by members of the Glut (or SLC2a) family of membrane glycoproteins that includes 14 known transporters. The Glut family belongs to the Major Facilitator Superfamily (MFS), which comprises the largest group of membrane transport proteins. The Gluts are responsible for the exchange of glucose between the blood and the cytoplasm of cells, supplying glucose for energy metabolism and biosynthetic reactions. Additionally, glucose transport in certain tissues plays a critical role in whole body glucose homeostasis and is associated with several disease states, including type 2 diabetes, Glut1-deficiency syndrome, Fanconi-Bickel syndrome, tumor progression, and cellular invasion by Human T-Cell Leukemia Virus. Despite the physiologic and clinical importance of glucose transport, relatively little is known concerning the structure and function of the Glut proteins. Recently, high-resolution structures were reported for two bacterial members of the MFS, which has enabled the development of a low-resolution structural model for Glut1 based on homology modeling. The long-term goals of this project are to delineate the structure of Glut1 and the molecular mechanism of facilitative sugar transport. To make further progress towards accomplishing these long-term goals, the following specific aims are proposed for the next project period: 1. Nine of the 12 predicted transmembrane helices and approximately 41% of the 492 residues comprising Glut1 have been analyzed thus far by site-directed mutagenesis. The 3 remaining transmembrane helices will be analyzed by cysteine-scanning mutagenesis in conjunction with the substituted cysteine accessibility method (SCAM). 2. Proximity relationships between pairs of transmembrane helices will be determined by chemical crosslinking of di-cysteine mutants constructed in novel functional cysteine-less Glut1 reporter constructs. 3. Procedures will be developed for the purification of milligram quantities of recombinant Glut1 and Glut4 proteins in an attempt to produce crystals for X-ray diffraction analysis. This aim will be conducted in collaboration with Dr. Da-Neng Wang at NYU.